Modeling and Rescue Strategies for Ciliopathies using Stem Cells Ciliopathies include a spectrum of phenotypes with defects in primary cilia biogenesis and/or function. Many retinal diseases are characterized by defects in cilia including, but not limited to LCA and JSRD. CEP290 is a gene which is important for cilia biogenesis and transport. Ciliary functions of patients can be differentially affected due to distinct mutations in this gene, leading to CEP290-related ciliopathies. Therefore, understanding the molecular mechanisms underlying the retinal development in JSRD and LCA patients is the utmost need for the development of treatments for these diseases. We attempted to model LCA and JSRD pathogenesis and explore different therapeutic interventions using fibroblasts of mouse model, i.e., rd16, and patients, as well as iPSC-derived 3-D retinal organoids. We showed that both rd16 mouse embryonic fibroblasts and organoids demonstrate defects in ciliogenesis. In collaboration with our NEI colleagues, we demonstrated that this phenotype could be rescued by delivering the myosin-tail domain of CEP290 protein. Using human iPSC lines from fibroblasts of LCA and JSRD patients and their familial controls, we have generated retinal organoids from these iPSC lines and collected neural retina at different stages of differentiation for immunohistochemistry, transmission electron microscopy and transcriptome analysis. We have shown that CEP290-LCA patient iPSC-derived photoreceptors displayed defects in ciliogenesis, including docked mother centrioles and membraneless intracellular microtubules. With our modified protocol for human retinal organoid culture, these photoreceptors displayed many disease relevant phenotypes, with failure to develop outer-segment like structure and mis-localization of Rhodopsin. Our studies thus recapitulate the pathologic changes in CEP290-LCA patients and should serve as a useful model to test treatment strategies. We have also generated iPSCs lines from fibroblasts of JSRD patients and familial controls with mutations within a cilia related gene, NPHP5. Retinal organoids have been generated with morphology similar to positive control retinal organoids. We have recently started a similar strategy to study NPHP5 ciliopathy patient cells. In parallel, we are developing a Sonic hedgehog signaling assay to study the early defects in this cell line. Drug Discovery and Small Molecule Screening Using Retinal Organoids In attempt to find candidate drugs to rescue ciliopathy-related photoreceptor dysfunction, we took advantage of the short differentiation time of mouse retinal organoids and developed in vitro screening platforms using rd16 iPSC-derived organoids with mutations in CEP290. Defects in ciliogenesis resembling in vivo mouse retina was observed rd16 organoids and they subsequently led to dysfunction or death of rod photoreceptors in rd16 organoids. We performed high-throughput screenings of candidate drugs in collaboration with the National Center for Advancing Translational Sciences (NCATS) in an attempt to maintain survival of retinal organoid-derived photoreceptors. Our preliminary screening resulted in 30 positive hits. We are now validating these positive hits in vivo in mouse retina and in patient-derived retinal organoids. Modeling LCA caused by CRX mutations Photoreceptor dysfunction characteristic of patients with photoreceptor genetic disorder can be also caused by mutations in genes unrelated to cilia. One causal mutation of LCA is noted in CRX which encodes an essential transcription factor, cone-rod homeobox protein (CRX), for cone and rod development and function. To better understand the disease phenotype, we have derived human iPSCs from CRX-LCA patients and familial controls and differentiated them toward photoreceptors. To date, retinal organoids of both patient and control lines have been formulated. We have observed lamination similar to positive control retinal organoids indicating that differentiation is proceeding successfully. Future work will compare the transcriptome of CRX-LCA retinal organoids and controls to healthy embryonic tissue. Using a similar high-throughput system outlined above, candidate drugs to rescue disease phenotype will be screened. Analyzing transcriptome of retinal organoids derived from familial control iPSCs The use of iPSCs has provided a scientific opportunity to study rare retinal diseases where good animal models do not exist. This technology has been leveraged by multiple laboratories around the globe, however, protocols, efficiency, and success rate are highly variable. To elucidate the role of these differences, the transcriptomes of retinal organoids derived via 3 protocols, 3 individuals, and 4 pluripotent stem cell lines (1 embryonic and 3 control iPSC)vare being analyzed. Early results indicate that there are differences in differentiation rate of various organoids as compared to previously published fetal retina and adult data. Comparing the lines and differentiation protocols indicates that we have successfully developed an optimized protocol to accelerate formation outer segment-like structures. In Vitro 3-D Models of Retinal Diseases Modelling of retinogenesis in vitro has been hampered by limited development of outer segments essential to detection of light by photoreceptors. In collaboration with Tiansen Li, PhD, we hypothesize that a retinal pigmented epithelial (RPE) layer is necessary to form mature connecting cilia and functional outer segments. We have used an embryonic stem (ES) cell line (H9) and patient induced pluripotent stem cell lines (hiPSCs) to differentiate stem cells into RPE-like cells within 90 days of induction. Our differentiated RPE cells correctly express proteins similar to mature RPE cells, have polarity typical of RPE cells, and display distinct RPE morphology. This indicates that we can successfully differentiate stem cells lines toward RPE phenotypes with our protocol in a reliable manner. Our lab has also developed an number of electrospun or electroblown scaffolds that mimic the properties of the supporting structure of RPE, the Bruchs Membrane. To date, we have formed scaffolds composed of poly(caprolactone) (PCL), soy protein, and a collagen/elastin blend. Stem cells have been differentiated into RPE on these electrospun scaffolds and maintained similar characteristics of cells differentiated with our standard protocol. Initial results from our lab show stem cells differentiated on PCL-electrospun scaffolds develop markers for RPE, express necessary RPE proteins, and undergo morphological changes such as: hexagonal shape formation and polarization typical of RPE. We hypothesize that differentiating toward RPE cells on Bruchs Membrane mimics will increase efficiency and efficacy. Future work will test this hypothesis by comparing the transcriptome to embryonic human samples. Due to limited outer segment and cilia formation in vitro, functional assays for testing photoreceptor retina viability and functionality are unable to be performed. This limits the potential for patient- derived retinal organoids in patient-specific drug screening in vitro. We have begun to develop a co-culture model system with optic vesicles and differentiated RPE on electrospun scaffolds.